(The FASEB Journal. 1999;13:S113-S120.)
© 1999 FASEB
Electron microscopic analysis of gravisensing Chara rhizoids developed under microgravity conditions
MARKUS BRAUN1,
BRIGITTE BUCHEN and
ANDREAS SIEVERS
Botanisches Institut, Universität Bonn, Bonn, Germany
1Correspondence: Botanisches Institut, Universität Bonn, Venusbergweg 22, D-53115 Bonn, Germany. E-mail: unb13a{at}uni-bonn.de
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ABSTRACT
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Tip-growing, unicellular Chara rhizoids that react
gravitropically on Earth developed in microgravity. In microgravity,
they grew out from the nodes of the green thallus in random
orientation. Development and morphogenesis followed an endogenous
program that is not affected by the gravitational field. The cell
shape, the polar cytoplasmic organization, and the polar distribution
of cell organelles, except for the statoliths, were not different from
controls that had grown on earth (ground controls). The ultrastructure
of the organelles and the microtubules were well preserved.
Microtubules were excluded from the apical zone in both ground controls
as well as microgravity-grown rhizoids. The statoliths (vesicles
containing BaSO4 crystals in a matrix) in
microgravity-grown rhizoids were spread over a larger area (up to 50
µm basal to the tip) than the statoliths of ground controls (10–30
µm). Some statoliths were even located in the subapical zone close to
microtubules, which was not observed in ground controls. The crystals
in statoliths from microgravity-grown rhizoids appeared more loosely
arranged in the vesicle matrix compared with ground controls. The
chemical composition of the crystals was identified as
BaSO4 by X-ray microanalysis. There is evidence that the
amount of BaSO4 in statoliths of rhizoids developed in
microgravity is lower than in ground controls, indicating that the
gravisensitivity of microgravity-developed rhizoids might be reduced
compared with ground controls. Lack of gravity, however, does not
affect the process of tip growth and does not inhibit the development
of the structures needed for the gravity-sensing machinery.—Braun, M.,
Buchen, B., Sievers, A. Electron microscopic analysis of gravisensing
Chara rhizoids developed under microgravity
conditions.
Key Words: gravitropism • statolith • tip growth
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INTRODUCTION
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IN ADDITION TO the genetic information, perception of
and response to environmental stimuli are essential for development and
morphogenesis of organisms. Gravity is one important stimulus that
plants use to orient their growth direction and movement of their
organs. Gravity-sensing mechanisms that involve intracellular
sedimenting particles and their interaction with cytoskeletal elements
are common in plant cells and organs (1)
.
The unicellular, gravitropically tip-growing rhizoids of the green
alga Chara have been established as a model system for the
investigation of gravitropic mechanisms and cytological and functional
aspects of the plant cytoskeleton (1-3)
. In
downward-growing rhizoids on Earth, vesicles that contain
BaSO4 crystals (4)
are located 10–30 µm
above the apical cell wall. These vesicles have been identified as
statoliths by basipetal centrifugation, which removes the vesicles from
their natural site in the apical zone and abolishes gravitropic
curvature when the rhizoids are tilted from the vertical
(5
, 6
). In downward-growing rhizoids, actin
microfilaments prevent the sedimentation of statoliths onto the apical
cell wall. Treatment of vertically growing rhizoids with cytochalasin D
disrupts the actin microfilaments and results in a sedimentation of
statoliths into the tip and termination of tip growth (7)
.
During the 6-min microgravity period of a
TEXUS2 rocket flight, the
statoliths were displaced basipetally and nearly doubled their original
distance from the apical cell wall (8)
. This was the first
in vivo videomicroscopic observation of such a microgravity
effect in a single cell. It was concluded that in rhizoids growing
downward at 1 g the statoliths are kept in a dynamically
stable position by the counteraction of two forces, gravity and
internal forces mediated by the acto-myosin system
(8-10)
.
When rhizoids were placed horizontally at 1 g before launch
of the TEXUS rocket, the statoliths sedimented on the lower cell flank.
Thereafter, in microgravity, their displacement was smaller in the
lateral direction (toward the former upper flank) than in the axial
direction (basipetally). Thus, the position of statoliths is highly
controlled and regulated in both axial directions but only weakly
controlled in the lateral direction (3)
. Optical tweezers
experiments showed the same results by measuring the laser output power
necessary to translocate statoliths in axial and lateral directions
(11)
. Both experiments and the exclusion of microtubules
from the apical zone (12
, 13
) suggest that
gravitropic tip growth in Chara rhizoids is dependent on a
highly polarized actin filament system that actively controls the
position of the statoliths in the axial direction and allows their
lateral sedimentation and the subsequent gravitropic curvature of the
cells (12
, 13
).
To understand the molecular mechanisms of the gravitropic response
chain, the detailed analysis of the interaction between the
cytoskeleton, the statoliths, and the apical tip-growth-organizing
complex, the Spitzenkörper, is essential not only at 1
g but also under microgravity, which provides an almost
stimulus-free environment. During the IML-2 SpaceHab mission rhizoids
were fixed for electron microscopy under long-term microgravity
conditions for the first time (14)
and the first data on
gravisensitivity were obtained. The rhizoids grown in microgravity
revealed the same polar distribution of organelles and structural
organization as in ground controls. However, these cells had developed
at 1 g on ground before launch and the electron-microscopic
images delivered no information on the cytoskeleton and the apical
aggregation of endoplasmic reticulum (ER), which is argued to represent
the structural center of the Spitzenkörper (3)
. The
Chara Biorack experiments during the S/MM-05 SpaceHab
mission therefore had been designed to investigate the ultrastructure
of rhizoids that have developed and grown exclusively under
microgravity conditions. In addition, a fixation method should be
tested that would allow embedding of cells in a hydrophilic medium (LR
White) for detection and localization of cytoskeletal epitopes by
immunolabeling.
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MATERIALS AND METHODS
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Green thalli of Chara globularis Thuill. were
collected from a pond at the Botanischer Garten (Universität
Bonn) and cultured at Hangar L, Kennedy Space Center, Florida. Culture
of rhizoids has already been described (14)
.
For all flight and ground experiments, the culture chambers containing
thallus segments with very short rhizoids (set A) or no visible
rhizoids (set B) were inserted into the fixation units that were housed
in the Biorack type 1 container (Dornier, Friedrichshafen, Germany)
adapted for illumination of the algae by the insertion of four windows.
The construction of the fixation unit and the fixation procedure has
been described in detail (14)
.
The rhizoids of set A grew 18 h in the culture chambers in
darkness at 1 g before launch and for 46 h in
microgravity at SpaceHab ambient temperature (22 ± 1°C) until
fixation. In the culture chamber of set B, rhizoids developed in
microgravity for 98 h until fixation. To provide light for the
rhizoid growth, two pouches containing 3 Biorack type 1 containers each
and one temperature recorder were fixed with Velcro tape close to the
SpaceHab lamps. Fixatives used were as follows: 0.5%
glutaraldehyde + 3% formaldehyde in microtubule-stabilizing
buffer (0.2 M PIPES, 5 mM EGTA, 5 mM MgSO4, pH 7.2) for two
chambers of set A and 3% glutaraldehyde in microtubule-stabilizing
buffer for one chamber of set A and all three chambers of set B. When
the exchange of culture medium by fixation solution was finished, the
valves were closed. The fixation unit was inserted into the Biorack
type 1 container and stored at 4°C in a cooler until landing at
Kennedy Space Center.
The embedding of samples in Spurr's resin was performed after landing
and followed the previously described procedure (14)
. For
immunogold labeling, samples of set A were washed in
microtubule-stabilizing buffer, followed by two washing steps with
phosphate-buffered saline (PBS). These samples were dehydrated with an
ethanol series and slowly infiltrated with LR White (Polysciences,
Inc., Warrington). The ultrathin sections were blocked with PBS
containing glycine, gelatine fish, and Tween-20, incubated in the first
(mouse anti-tyrosine 
-tubulin, Sigma T-9028; rabbit anti-actin,
Sigma A-2668) and gold-conjugated second antibodies for 2 h, and
postfixed with glutaraldehyde (2.5%) for 10 min. Sections were
postcontrasted with a mixture of potassium permanganate and uranyl
acetate (1:4). All samples were examined with an EM 10 transmission
electron microscope (Zeiss, Oberkochen, Germany). X-ray microanalysis
(EDAX) was performed with a Cambridge S200 scanning electron microscope
using LR White sections of the same thickness (100 nm) and microscope
settings.
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RESULTS
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Rhizoids developed and grew under microgravity conditions. Without
net acceleration, rhizoids emerged and radiated from the thallus nodes
randomly in all directions (Fig. 1
). Tip growth of the rhizoids in microgravity followed either a
relatively straight line, a wavy line due to bending and rebending, or
an arched line. Both glutaraldehyde and glutaraldehyde/formaldehyde
fixation and embedding in Spurr's resin or in the hydrophilic LR White
resin resulted in a high-quality preservation of the flight and ground
samples. The prolonged fixation time obviously had no negative effect
on the quality of the ultrastructural preservation. On the
light-microscopic level, the rhizoids of ground controls, set A
(ground-developed and microgravity-grown), and set B
(microgravity-developed and -grown rhizoids) revealed the same cell
shape and the same polar cytoplasmic organization. The distance of the
nucleus from the cell tip in most rhizoids was in the normal range of
ground controls (250–300 µm). There was also no obvious difference
in the rates of tip growth (90–140 µm/h) estimated by the total
length of the rhizoids (Fig. 1)
.
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Figure 1. Micrograph of a Chara thallus node from which rhizoids
emerged and grew in all directions aboard the Space Shuttle Atlantis
under microgravity conditions. Diameter of the rhizoids is 30 µm.
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On the electron microscopic level, the distribution of organelles in
all zones of microgravity-developed and -grown rhizoids was the same as
in ground controls. The dimension of the cell wall, the size of the
nucleus (including the nucleolus), the large basal vacuole, the
relatively stationary cytoplasm of the subapical zone and of the apical
zone of microgravity-developed rhizoids were in the normal range of
ground controls. The same applies for the randomly distributed
organelles (mainly plastids and mitochondria) in the layer of streaming
cytoplasm that reverses its direction between the nucleus and the
apical end of the vacuole (Fig. 2
). The thickness of the basal cytoplasmic layer was the same as in
ground controls, indicating that an artificial deformation or swelling
had not been produced. The cytoplasm remained well attached to the
basal cell wall, which was also a good indication for the fast,
successful fixation. It can be concluded that the movement of the
cytoplasmic streaming was stopped quickly without generating turbulence
or bubbles. The large vacuole did not collapse or disintegrate. The
tonoplast, however, was partially disrupted or broken, a feature that
was also observed in ground controls and thus was not specific or
significant for samples grown in microgravity.
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Figure 2. Electron micrograph of a part of the nucleus and the apical end of the
large basal vacuole (V) from a rhizoid developed under microgravity
conditions. The structural integrity of the nucleus (N), nucleolus
(N'), and the organelles are well maintained and the membranes are
well preserved. Fixation was fast enough to stabilize the layers of
ecto- and streaming endoplasm between the vacuole and the cell wall.
Bar = 5 µm.
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Structure and integrity of the organelles like mitochondria,
proplastids, dictyosomes, secretory vesicles (Golgi vesicles), and
microvesicles was similar to that of ground controls (Figs. 3-5
).Dictyosomes were observed in different orientations with well-preserved
cis- and trans-regions and the fenestrated
trans-Golgi network with an abundance of vesicles (Fig. 3)
.
ER cisternae were oriented predominantly parallel to the length axis of
the cell (Figs. 3-5)
. The cisternae were especially well preserved
(not vesiculated) and the ribosomes were clearly recognizable.
Numerous, mainly axially oriented, microtubules were observed in
peripheral and median sections of the subapical zone (Fig. 3-5)
.
Immunolabeling of microtubules was for the first time successfully
performed in LR White sections of microgravity-grown rhizoids with the
use of gold-conjugated second antibodies (Fig. 5
, inset); the epitopes
were still recognized by the antibodies even after the prolonged
fixation time.
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Figure 3. Detail of the subapical zone of a microgravity-developed
Chara rhizoid showing dictyosomes (D) with clearly
discernible stacks of cisternae, the cis- and
trans-Golgi-regions, the fenestrated trans-Golgi
network (arrow), and abundant vesicles. Bar = 1 µm.
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Figure 5. Microtubules (MT) in longitudinal sections (Spurr's resin) of the
subapical zone of a Chara rhizoid developed in microgravity.
Microtubules and endoplasmic reticulum cisternae (ER) are oriented
parallel to the cell axis as was also found in ground controls.
Bar = 0.2 µm. Inset: immunogold detection of a microtubule (LR
White section). Diameter of the gold particles = 10 nm.
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Microtubules had never been detected in the apical zone of control
rhizoids at 1 g and they were also not present in the apex
of rhizoids that had developed and grown in microgravity (Figs. 6
and
7).In the apical cytoplasm of flight samples the dense aggregation of ER
membranes in the center of the Spitzenkörper was also well
preserved surrounded by a great number of secretory vesicles and
microvesicles (Fig. 6)
. Plastids, mitochondria, and dictyosomes were
only sporadically present in the apical zone.
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Figure 6. Electron micrograph showing a part of the apical zone of a
Chara rhizoid developed in microgravity. A great number of
secretory vesicles (SV), microvesicles (MV), and the aggregation of
endoplasmic reticulum (ER) forming the Spitzenkörper of the
rhizoid are well preserved by the glutaraldehyde fixation performed in
microgravity in the Biorack glovebox. Bar = 1 µm.
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Thin filament-like structures have been detected in the apical and
subapical zone of Spurr's resin sections (Fig. 7)
. These filaments
have a diameter of approximately 10 nm and were found in various
orientations but in the apical dome they were predominantly running
toward the apical membrane. Numerous vesicles appeared to be lined up
along these filaments. Gold-conjugated antibodies against actin
epitopes occasionally labeled short filament-like structures in LR
White sections; however, the labeling was scarce and not consistent in
all sections analyzed. This was also the case in ground controls.
Therefore, there are some indications that actin is present in the very
tip of flight samples as shown by immunolabeling and
rhodamine-phalloidin labeling in rhizoids of ground samples
(13)
; however, further studies are needed for proof.
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Figure 7. Electron microscopic detail of the apical zone at the outermost tip of
a Chara rhizoid developed in microgravity. Putative actin
microfilaments are present in the apical cytoplasm in the form of thin
filament-like structures (arrows). Microvesicles (arrowheads) are lined
up along these filaments. Inset: possible immunogold detection (LR
White section). Bar = 0.2 µm.
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In contrast to ground controls, the statoliths in
microgravity-developed and -grown rhizoids were located at a distance
of 5–50 µm from the cell tip. Accordingly, some statoliths were
found close to microtubules in the subapical zone (Fig. 4
and
Fig. 8
).In vertically downward growing rhizoids at 1 g, however,
statoliths are positioned at a distance of 10–30 µm basal to the
cell tip and they have never been observed close to microtubules.
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Figure 4. In microgravity-developed rhizoids, the general arrangement and
orientation of the subapical organelles were the same as in ground
controls. ER cisternae (ER) are mostly oriented parallel to the cell
wall. Plastids (P), dictyosomes (D), numerous microvesicles, and
vesicles of different size and contrast are randomly distributed
throughout the subapical cytoplasm. An individual statolith (St) is
located close to a microtubule (arrow) at the apical end of the
subapical zone. Bar = 1 µm.
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Figure 8. A statolith is located close to microtubules (arrows) in the subapical
zone, a localization that does not occur in normal vertically oriented
rhizoids at 1 g. Bar = 0.5 µm.
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Differences were also found in the ultrastructure of statoliths from
flight and ground samples. In rhizoids grown at 1 g, seven
of nine statoliths are filled homogeneously with well-contrasted
BaSO4 crystals in a matrix of medium electron density
(Fig. 9A
).In contrast, the matrix of 14 of 17 statoliths in the flight samples
was less electron dense and showed either a gradient of highly
contrasted crystals from the center to the periphery or a lack of
crystals in the cortical (Fig. 9B
) and/or central region of
the statoliths (Fig. 9C
). These structural features and
differences were analyzed in median-sectioned statoliths.
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Figure 9. Micrographs showing statoliths from rhizoids grown at 1 g
(A) and in microgravity (B, C). At 1
g, statoliths have a dense matrix, homogeneously filled with
BaSO4 crystals, whereas the statoliths developed in
microgravity contain a less dense matrix and a BaSO4
crystal-free space in the cortical (B) or in the inner
(C) region. Bar = 0.5 µm.
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X-ray microanalysis of LR White-embedded statoliths confirmed that
microgravity-developed rhizoids also contained crystals of
BaSO4. It is interesting to note, however, that
quantitative analyses indicated a reduction in the amount of barium and
sulfur in all statoliths analyzed from microgravity-developed and
-grown rhizoids (n = 6) compared with ground controls
(n = 5; Fig. 10
).The signals from cytoplasmic areas outside the statoliths can be
considered as internal controls; neither barium nor sulfur was
detected. Due to the fixation procedure, soluble ions like potassium
cannot be taken as additional parameters for quantification of
elements; these elements varied in the height of the signals in all
spectra. To enable a relative comparison of the insoluble crystal
compounds, the X-ray energy-dispersive spectra had been made from
sections of rhizoids identically fixed and embedded and of the same
thickness. We also used the same microscope set-up and recorded data
with the same count rates.
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Figure 10. EDAX spectra of statoliths from ground controls (1 g) and
microgravity-developed rhizoids (µG) demonstrating the reduction of
barium and sulfur in microgravity-developed statoliths. A reference
spectrum was taken from the subapical zone of microgravity-developed
Chara rhizoids (R).
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DISCUSSION
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The fixation and ultrastructural analysis of rhizoids, which have
developed and grown exclusively under microgravity conditions,
confirmed the results obtained with rhizoids that had developed on
ground and continued growth in microgravity during the IML-2 mission
(14)
. The tip-growing rhizoids followed their endogenous
program for development and morphogenesis even in the absence of net
acceleration in microgravity. Neither the cell shape nor the polar
cytoplasmic zonation were different from ground controls. Due to the
absence of a directing acceleration force, the rhizoids emerged and
grew out from the nodes in all directions. Nevertheless, rhizoids are
adapted to sense the direction of gravity and reorient in the
gravitational field and it has already been demonstrated that rhizoids,
which never or only for a very short time had experienced a
considerable gravitational field, had not lost the ability to respond
to gravity (3)
.
The polar distribution of organelles was also established in
microgravity-developed rhizoids. The unaltered cell shape and growth
rates and the presence of the ER aggregate in the center of the
Spitzenkörper, including secretory vesicles and microvesicles,
suggest that the process of tip growth was not affected by the absence
of gravity or by the conditions of spaceflight. The well-preserved
tubular ER membranes that form a dense aggregation in the apex of
rhizoids have been shown in microgravity-grown rhizoids for the first
time and differ considerably from the axially oriented ER membranes in
the subapical zone. It has been argued that they might be involved in
the process of gravitropic tip growth, most likely by maintaining and
regulating the tip-high calcium gradient in the apical dome
(3)
.
The fixation for LR White embedding and immunogold labeling was
successfully performed in microgravity. Even after the prolonged
fixation time and storage in the SpaceHab cooler under microgravity
conditions, the antigenicity was still sufficient for immunogold
labeling of the microtubule cytoskeleton. The labeling and localization
of microtubules in ultrathin LR White sections of
microgravity-developed rhizoids was in accordance with immunogold
labeling and immunofluorescence labeling of microtubules in rhizoids
grown at 1 g (3
, 12
,
13
). Microtubules were well preserved and localized in the
basal zone and the subapical zone but not in the apical zone. The
exclusion of microtubules from the apical zone indicates that the polar
arrangement of the microtubule cytoskeleton was maintained in
microgravity. Microtubules play a crucial role in stabilizing the polar
cytoplasmic zonation, the position of the nucleus and the organelles,
as well as the fine netlike arrangement of the actin microfilaments in
the subapical zone, but they are not involved in the primary steps of
graviperception and graviresponse (12)
. The absence of
apical microtubules is a precondition for the fast and unimpeded
sedimentation of statoliths on the physically lower cell flank and the
following graviresponse (12)
.
Fine actin microfilaments form a dense meshwork in the subapical zone
and converge in a bright apical spot, as was demonstrated by
immunofluorescence and rhodamine-phalloidin labeling (13)
.
The actin spot coincides spatially with the position of the ER
aggregate, which is organized and positioned by the actin cytoskeleton.
Actin microfilaments also organize and coordinate the process of tip
growth (13
, 15
). In microgravity-developed
rhizoids, thin filament-like structures were detected in the subapical
and apical zone with a number of microvesicles lined up along these
filaments. The correlation of the actin fluorescence in the rhizoid
apex (1
, 2
, 3
, 9
,
13
) with the ultrastructure and the dimension of these
filaments suggests that they might represent actin microfilaments. The
difficulties in preserving and stabilizing actin microfilaments without
reducing their antigenicity are well known (15)
and we
cannot draw conclusions on the overall arrangement of the actin
cytoskeleton. However, the ultrastructural findings and the unaltered
process of tip growth of rhizoids grown in microgravity indicate no
major changes in the functional and structural arrangement of the actin
cytoskeleton compared with rhizoids grown at 1 g.
Statoliths were found in similar number and size in
microgravity-developed rhizoids as in ground controls. In
microgravity-developed rhizoids, the statoliths were similarly
distributed as in rhizoids after only 6 min of microgravity during the
parabolic flight of a TEXUS rocket (1
, 8
).
Therefore, it can be concluded that a new dynamically stable position
of statoliths is achieved early on, does not change during hours of
growth in microgravity, and is similar in rhizoids that have developed
in microgravity. However, it is noteworthy that only the position of
the statoliths at 1 g, which is determined by actin
microfilaments and gravity, is optimal for the gravitropic curvature.
Only statoliths positioned in the apical zone can sediment quickly on
the lower cell flank and initiate graviresponse (6)
.
Sedimentation of statoliths positioned further basally in the subapical
zone is strongly impeded (12)
.
According to Kiss (16)
, the gravitropic curvature is
correlated with the number of statoliths. Thus, even with a small
number of statoliths, rhizoids are still able to respond to gravity;
they are, however, perfectly adapted to the Earth's gravitational
field. In consequence, a decrease of the BaSO4 content in
the statoliths of microgravity-grown rhizoids should result in a longer
sedimentation time and an enhanced threshold value of gravisensitivity.
A reduction in the density of statoliths under microgravity has also
been reported for other cell types; i.e., the amount of starch was
reduced in the statoliths of higher plant statocytes (17
,
18
) and the BaSO4 content in Müller
bodies of the ciliate Loxodes was also reported to be lower
than in ground controls (19)
. It can be excluded that the
structural differences between statoliths of ground- and
microgravity-grown rhizoids result from an artifact produced by the
fixation as is the case with KMnO4 fixation
(4)
. The absolute values of the statolith density of
microgravity-grown rhizoids or at least the difference in the amount of
BaSO4 induced by the change of the gravitational
environment must be determined in the future. The cytoskeleton itself
might also adapt to microgravity conditions and might affect the
threshold value. Adaptation of actin microfilaments has already been
reported during basipetal centrifugation of Chara rhizoids
(20)
. Threshold experiments with higher plant roots
indicated a higher gravisensitivity of microgravity-developed roots
based on cytoskeletal adaptation (21)
. Nevertheless, both
the reduction of the statolith's mass (this study and refs.
17-19
) and the adaptation of the cytoskeleton
(20
, 21
), affect the gravisensing mechanism.
Therefore, it should be taken into account that the gravisensitivity of
microgravity-grown rhizoids might somewhat differ from ground controls,
at least in cells grown for a longer period in a hyper- or hypogravity
environment; the same might also apply for other biological
systems.
|
ACKNOWLEDGMENTS
|
|---|
This work was supported by the AGRAVIS project of the Deutsches
Zentrum für Luft und Raumfahrt, Bonn, and Ministerium für
Wissenschaft und Forschung, Düsseldorf. The authors thank the
European Space Agency Biorack team (Nordwijk, Netherlands), the Dornier
team (Friedrichshafen, Germany), the Bionetics team at Hangar L, and
the NASA team (Kennedy Space Center) for their assistance and skillful
support. Special thanks to the crew members of IML-2 and S/MM-05 for
carrying out the experiments. We thank Simone Masberg for excellent
technical assistance and Hans-Jürgen Ensikat for his help with
EDAX.
|
FOOTNOTES
|
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2 EDAX, energy dispersive X-ray microanalysis;
IML-2, international microgravity laboratory; S/MM-05 shuttle to
Mir mission; TEXUS, technological experiments under
microgravity; PBS, phosphate-buffered saline; ER, endoplasmic
reticulum.
Received for publication November 12, 1998.
Revision received January 15, 1999.
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[Abstract]
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ABSTRACT
INTRODUCTION
